GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus

Background: Detection and quantification of human papillomavirus (HPV) may help in predicting the evolution of HPV infection and progression of associated lesions. Objectives: We propose a novel protocol using consensus primers GP5+/6+ in a SYBR Green quantitative real-time (Q-RT) polymerase chain reaction (PCR). The strategy permits screening for HPV infection and viral load quantification simultaneously. Study design: DNA from 153 archived cervical samples, previously tested for HPV detection by GP5+/6+ PCR and typed by EIA-RLB (enzyme immunoassay-reverse line blot) or sequence analysis, was analysed using SYBR Green Q-RT PCR. Melting temperature assay (T(m)) and cycle threshold (C(t)) were used to evaluate HPV positivity and viral load. The T(m) in the range of 77-82 degrees C was considered to be positive for HPV-DNA. HPV results generated through GP5+/6+ conventional PCR were considered the gold standard against which sensitivity and specificity of our assay were measured. Results: Out of 104 HPV positive samples, 100 (96.2%) were also determined as positive by SYBR Green Q-RT PCR; of the 49 HPV-negative samples, all were determined as negative. There was an excellent positivity agreement (K = 0.94) between the SYBR Green Q-RT and the previous methods employed. The specificity and sensitivity were 100% and 96.2%, respectively. Comparison of SYBR Green Q-RT and TaqMan oligo-probe technologies gave an excellent concordance (pc = 0.95) which validated the proposed strategy. Conclusions: We propose a sensitive and easy-to-perform technique for HPV screening and viral load quantification simultaneously. (C) 2009 Elsevier B.V. All rights reserved.

Efficiency of delivery of DNA to cells by bovine papillomavirus type-1 L1/L2 pseudovirions

To investigate the efficiency of encapsidation of plasmid by papillomavirus virus-like particles (PV VLPs), and the infectivity of the resultant PV pseudovirions, Cos-1 cells were transfected with an 8-kb plasmid incorporating a green fluorescent protein (GFP) reporter gene (pGSV), and infected with bovine PV (BPV-1) L1/L2 recombinant vaccinia virus to produce BPV1 pseudovirions. Approximately 1 in 1.5x10(4) of dense (1.35 g/ml) PV pseudovirions and 0.3 in 10(4) Of less-dense (1.29 g/ml) pseudovirions packaged an intact pGSV plasmid. The majority (>75%) of packaged plasmids contained deletions, and the deletions affected all tested genes. After exposure of Cos-1 cells to BPV-1 pseudovirions at an MOI of 40,000:1, 6% of cells expressed GFP giving a calculated efficiency of delivery of the pGSV plasmid, by pseudovirions which had packaged an intact plasmid, of approximately 5%. Plasmid delivery was not effected by purified pGSV plasmid, was blocked by antiserum against BPV-1, and was not blocked by DNase treatment of pseudovirions, confirming that delivery was mediated by DNA within the pseudovirion. We conclude that a major limitation to the use of PV pseudovirions as a gene delivery system is that intact plasmid DNA is not efficiently selected for packaging by VLPs in cell-based pseudovirions production systems.

Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions

Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy. S. cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S. cerevisiae. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S. cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect S. cerevisiae and that BPV-1 episomes can replicate in the infected S. cerevisiae cells.

Saccharomyces cerevisiae is permissive for replication of bovine papillomavirus type 1

We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derived DNA. Virus particles prepared in S. cerevisiae are able to convey packaged DNA to Cos1 cells and to transform C127 cells. Infectivity was blocked by antisera to BPV1 L1 but not antisera to BPV1 E4. We conclude that S. cerevisiae is permissive for the replication of BPV1 virus.

Hybrid capture II and PapilloCheck® tests for detection of anal high-risk human papillomavirus

Introduction This study evaluated the level of concordance between hybrid capture II (HCII) and PapilloCheck® for the detection of high-risk human papillomavirus (HPV) in anal samples. Methods Anal cell samples collected from 42 human immunodeficiency virus (HIV)+ patients were analyzed. Results Considering only the 13 high-risk HPV types that are detectable by both tests, HCII was positive for 52.3% of the samples, and PapilloCheck® was positive for 52.3%. The level of concordance was 80.9% (Kappa = 0.61). Conclusions Good concordance was observed between the tests for the detection of high-risk HPV.

Human papillomavirus (HPV) screening and cervical cancer burden. A Brazilian perspective

This review tackles the issues related to disease burden caused by cervical cancer (CC) and its precursor (CIN) lesions in Brazil. A special focus is given to new technologies with potential to interfere with the development of CC by reducing the high-risk human papillomavirus (hr-HPV)-induced lesions that remain a major public health burden in all developing countries where organized screening programs do not exist. Globally, 85 % of all incident CC and 50 % of CC deaths occur in the developing countries. Unfortunately, most regions of Brazil still demonstrate high mortality rates, ranking CC as the second most common cancer among Brazilian women. Recently, CC screening programs have been tailored in the country to enable early detection of CC precursor lesions and thereby reduce cancer mortality. A combination of HPV testing with liquid-based cytology (LBC) seems to be a promising new approach in CC screening, with high expectation to offer an adequate control of CC burden in this country.

Immune response in cervical dysplasia induced by human papillomavirus: the influence of human immunodeficiency virus-1 co-infection - review

Human immunodeficiency virus (HIV-1) has become an important risk factor for human papillomavirus (HPV) infection and the development of HPV associated lesions in the female genital tract. HIV-1 may also increase the oncogenicity of high risk HPV types and the activation of low risk types. The Center for Disease Control and Prevention declared invasive cervical cancer an acquired immunodeficience virus (AIDS) defining illness in HIV positive women. Furthermore, cervical cancer happens to be the second most common female cancer worldwide. The host's local immune response plays a critical factor in controlling these conditions, as well as in changes in the number of professional antigen-presenting cells, cytokine, and MHC molecules expression. Also, the production of cytokines may determine which arm of the immune response will be stimulated and may influence the magnitude of immune protection. Although there are many studies describing the inflammatory response in HPV infection, few data are available to demonstrate the influence of the HIV infection and several questions regarding the cervical immune response are still unknown. In this review we present a brief account of the current understanding of HIV/HPV co-infection, emphasizing cervical immune response.